PROJECT SUMMARY Ovarian cancer is the leading cause of deaths due to gynecological malignancies in women and will accounts for over 14,000 deaths in the United States this year alone. However, current therapies do not provide meaningful long-term clinical benefits to ovarian cancer patients. Therefore, new and more effective therapies for ovarian cancer treatment are urgently needed. We found that ovarian cancer cells epigenetically downregulate the Natural Killer Group 2 D (NKG2D) ligands that are necessary for Natural Killer (NK) cells of innate immune system to eradicate cancer cells. Based on these findings, we rationalized that an approach that will allow re-expression of NKG2D ligands will enhance NK cell-mediated eradication of ovarian cancer cells and will be of therapeutic value. To identify such epigenetic drug targets that when inhibited will increase the expression of NKG2D ligand in ovarian cancer cells, we performed a large-scale epigenome-wide shRNA screen and identified Disruptor of Telomeric Silencing 1-Like (DOT1L) as a regulator of NKG2D ligand ULBP1. We found that DOT1L inhibition by short-hairpin RNA (shRNA) or by a small-molecule inhibitor, EPZ-5676, significantly increased eradication of ovarian cancer cells by NK cells. Based on these results, we hypothesize that DOT1L by regulating the expression of NKG2D ligands regulates NK cell-mediated eradication of ovarian cancer cells. The overall objective is to rigorously determine the role of DOT1L as a driver of ovarian cancer tumor growth and progression that function in a NK cell-dependent manner and test the clinical value of pharmacologically inhibiting DOT1L for treating ovarian cancer. Aim 1 experiments will focus on determining the in vivo role of DOT1L as a driver of ovarian cancer growth and ascertain if this function of DOT1L is dependent on NK cells. To this end, based on our results in immunocompetent syngeneic mouse models, we will use a novel humanized mouse model with functional human immune system containing NK cells to test the DOT1L inhibition in a more human disease relevant pre-clinical mouse model setting. We will also determine the mechanism of DOT1L action. To this end, based on our preliminary results, we will test the role of NKG2D ligands and other potential mechanisms, such as regulation of ICAM1 by DOT1L, in regulating the ability of DOT1L on NK cell-mediated eradication of ovarian cancer. Aim 2 experiments will determine if pharmacological inhibition of DOT1L in vivo blocks ovarian cancer growth in a NK cell-dependent manner. To this end, we will determine if DOT1L inhibitor, EPZ-5676, inhibits ovarian tumor growth in a NK cell-dependent manner in humanized and in the pre-clinical patient-derived xenograft (PDX) models of ovarian cancer. Collectively, the results of our studies will identify a novel function of DOT1L in regulating NK cell activity against ovarian cancer cells in vivo and validate DOT1L small-molecule inhibitors to enhance NK cell function as a new therapeutic approach for treating ovarian cancer.!